Close association between DNA polymorphism of bovine major histocompatibility complex class I genes and serological BoLA-A specificities

Class I genes of the bovine major histocompatibility complex (MHC) were investigated by Southern blot hybridization and by serological analysis. A large number of class I restriction fragments and an extensive polymorphism were revealed when genomic DNA samples, digested with the restriction enzyme PvuII, were hybridized with a human cDNA probe. The result indicated the presence of multiple class I genes in cattle. The extensive restriction fragment length polymorphism (RFLP) was interpreted genetically by analysing five paternal half-sib families comprising, besides the bulls, 50 offspring and their dams. No less than 21 RFLP types were distinguished in this limited sample. The class I polymorphism was also analysed using a serological test with sera corresponding to four workshop specificities (w2, w6, w10 and w16) and three locally defined specificities (SRB1, SRB2 and SRB3). There was an excellent agreement between the two typing methods since no RFLP type was associated with more than one specificity and five of the seven specificities were associated with a single RFLP type. Evidence for close genetic linkage between class I and DQ class II genes was obtained since no recombinant was found among 45 informative offspring. Linkage disequilibrium among class I, DQ class II and C4 genes was also observed. The blood group specificity M' was completely associated with the w16 class I specificity and with the haplotype I1DQ1BC4(2) in this material.     

DNA polymorphism in the major histocompatibility complex of man and various farm animals

In the past few years it has been possible by combining enzymatic cleavage of genomic DNA and the Southern blot hybridization technique to explore the endonuclease recognition site polymorphism of the MHC. HLA class I and DR and DQ alpha and beta class II specific probes as well as human C4 and Bf class III probes were used. All these probes were shown to cross-hybridize with DNA from pigs, cattle, sheep and horses. Hybridization of human genomic DNA with a class I probe showed 15-25 bands per genome depending on the enzyme used. Distinct endonucleases generated clusters of restriction fragments (RF) in HLA-informative families which correlated with HLA specificities. While numerous clusters were found associated with HLA-A alleles almost no cluster was related to HLA B or C specificities. Similarly, class II probes provided a large number of clusters. The existence of these clusters suggested that some polymorphic restriction sites are found in strong linkage disequilibrium and that the underlying mechanism might be gene conversion with heteroduplex correction. Since the degree of polymorphism detected by RF appears to be greater than the polymorphism defined by more traditional methods stronger associations between RF and pathological conditions are to be expected. Southern blot analysis was applied to unrelated pigs and sheep, as well as to families. Preliminary studies have also been performed on a few unrelated cattle and horses. Depending on the endonuclease used the HLA class I probe hybridized with around 15 bands in MHC heterozygous pigs and ruminants while up to 20 bands were found in horses. Therefore, a several-fold greater number of potential class I genes exist compared to those actually expressed. With the class II beta probe, cattle and sheep showed around 10 bands whereas 15 were observed in pigs and around 20 in horses. Based on limited results obtained with DQ alpha and beta probes and with the DR alpha probe there appeared to be fewer of these respective genes. Only one C4 gene has been detected in pig and this gene maps within the SLA region. Hybridization with the human C4 probe in cattle, sheep and horses revealed two to four bands which could possibly account for two C4 genes. To date their linkage to the MHC has not been established. The Southern blot hybridization technique represents a powerful tool for future immunogenetic studies.(ABSTRACT TRUNCATED AT 400 WORDS)     

Investigation of genetic diversity in wild colonies of naked mole-rats (Heterocephalus glaber) by DNA fingerprinting

DNA from 20 individuals from four wild colonies of naked mole-rats, Heterocephalus glaber , were analysed for restriction fragment length polymorphism of class I major histocompatibility complex genes and minisatellite DNA, both of which have been shown to be highly variable between individuals in other species. The minisatellite probe employed in this study revealed limited polymorphism in the DNA of naked mole-rats, both within and between neighbouring colonies. Of the two class I major histocompatibility complex probes, both showed a lack of polymorphism within colonies, while one revealed a single difference in the restriction fragment pattern between one colony and the other three. This probe also revealed a possible variation in copy number of genes in some individuals. The low numbers of bands on the restriction fragment pattern also indicated that the naked mole-rat MHC I, in contrast to that of other mammalian species, may contain relatively few genes homologous to the class I major histocompatibility complex of the mouse. The absence of variability in naked mole-rat DNA in these normally highly polymorphic loci suggests that there may be little or no genetic diversity either within or between closely neighbouring colonies of naked mole-rats in the wild. The lack of polymorphism in the MHC I questions its possible role in individual odour recognition in this species of rodent.     

The relationship between ovine lymphocyte antigens and parasitological and production parameters in Romney sheep

The occurrence of ovine lymphocyte antigen (OLA) types in two flocks of New Zealand Romney sheep was examined in relation to resistance to nematode parasites [as judged by faecal egg counts (FEC)], plasma pepsinogen levels, liveweight and weight gains. A panel of OLA-typing antisera (SY 1-16) which determine class 1 MHC antigens of the sheep was used. The OLA combination SY 1a + 1b was found exclusively in Romney sheep of both flocks having below average FEC, but was present in low frequency (5%). In one flock, sheep possessing this antigen combination had consistently lower FEC from weaning to 1 year of age than sheep without this combination. SY 6 occurred significantly more frequently in above average FEC sheep and was associated with significantly higher FEC during secondary challenge infection. Plasma pepsinogen levels were significantly lower in those sheep possessing SY 2 or SY 3 but these OLA types were not associated with lower than average FEC. No OLA type was associated with above average weight gain but in one flock sheep with SY 1b and SY 1a + 1b had significantly lower weight gains between weaning and 6 months of age than sheep without these OLA types. A similar association was not found in the other flock. Sheep in one flock with SY 16 were significantly heavier than those without this antigen. No other associations between OLA types and liveweight were found.     

Class I major histocompatibility complex antigens and C4 concentrations in sheep plasma

Data are presented demonstrating that high concentrations of complement protein C4 in sheep plasma are associated with a particular class I OLA specificity. By way of contrast, a similar association could not be demonstrated between C3 plasma concentrations and OLA specificities. These data support the hypothesis that gene(s) determining C4 plasma concentrations are linked to the ovine MHC.     

DNA polymorphism in the major histocompatibility complex of man and various farm animals*

Summary. In the past few years it has been possible by combining enzymatic cleavage of genomic DNA and the Southern blot hybridization technique to explore the endonuclease recognition site polymorphism of the MHC. HLA class I and DR and DQ alpha and beta class II specific probes as well as human C4 and Bf class III probes were used. All these probes were shown to cross-hybridize with DNA from pigs, cattle, sheep and horses. Hybridization of human genomic DNA with a class I probe showed 15–25 bands per genome depending on the enzyme used. Distinct endonucleases generated clusters of restriction fragments (RF) in HLA-informative families which correlated with HLA specificites. While numerous clusters were found associated with HLA-A alleles almost no cluster was related to HLA B or C specificities. Similarly, class II probes provided a large number of clusters. The existence of these clusters suggested that some polymorphic restriction sites are found in strong linkage disequilibrium and that the underlying mechanism might be gene conversion with heteroduplex correction. Since the degree of polymorphism detected by RF appears to be greater than the polymorphism defined by more traditional methods stronger associations between RF and pathological conditions are to be expected. Southern blot analysis was applied to unrelated pigs and sheep, as well as to families. Preliminary studies have also been performed on a few unrelated cattle and horses. Depending on the endonuclease used the HLA class I probe hybridized with around 15 bands in MHC heterozygous pigs and ruminants while up to 20 bands were found in horses. Therefore, a several-fold greater number of potential class I genes exist compared to those actually expressed. With the class II beta probe, cattle and sheep showed around 10 bands whereas 15 were observed in pigs and around 20 in horses. Based on limited results obtained with DQ alpha and beta probes and with the DR alpha probe there appeared to be fewer of these respective genes. Only one C4 gene has been detected in pig and this gene maps within the SLA region. Hybridization with the human C4 probe in cattle, sheep and horses revealed two to four bands which could possibly account for two C4 genes. To date their linkage to the MHC has not been established. The Southern blot hybridization technique represents a powerful tool for future immunogenetic studies. This is even more so in large farm animals where for various reasons it is almost impossible to conduct certain types of investigation that are easily performed in rodents or in man. Although the data are still preliminary, they already extend our knowledge of the MHC in domestic animals far beyond what could have been reasonably anticipated using conventional methods.     

Polymorphism of bovine major histocompatibility complex (MHC) class I genes revealed by polymerase chain reaction (PCR) and restriction enzyme analysis

The polymorphic exon 2-exon 3 region of bovine major histocompatibility complex (MHC) class I genes was amplified by polymerase chain reaction (PCR) from genomic DNA samples with characterized class I polymorphism. The primers for amplification were designed in conserved regions at the borders of exons 2 and 3, based on all available cDNA sequences. The primers should, therefore, amplify most expressed class I genes, but may also amplify non-expressed class I genes. The PCR amplified class I gene fragments of 700 bp were characterized on the basis of restriction fragment length polymorphism (RFLP). The PCR-RFLP analysis of class I genes showed that the bands in each digestion could be classified as non-polymorphic, as shared between several bovine lymphocyte antigen (BoLA)-A types, or as specific to a single BoLA-A type. The same primers were then used for amplification of class I gene fragments from eight Sahiwal animals, a breed which originated in the Indian subcontinent. These studies showed that BoLA class I PCR-RFLP could be used to study class I polymorphism in family groups.     

Syrian hamster DNA shows limited polymorphism at class I-like loci

The class I gene products of the Syrian hamster major histocompatibility complex are unique in that they lack functionally detectable polymorphism. Mouse cDNA and hamster genomic probes were used to analyze the hamster class I gene family using genomic Southern hybridization. These studies revealed that the hamster possesses a complex class I multigene family and that it shares extensive sequence homology with the corresponding mouse sequences. Unlike the mouse, however, the Syrian hamster demonstrates only limited restriction endonuclease polymorphism in these genes. These results suggest that the lack of detectable polymorphism in this species is directly related to limited DNA polymorphism. The data presented here support the hypothesis that this species has undergone an evolutionary bottleneck, i. e., that all surviving members of the species arose from a limited number of progenitors.Abbreviations used in this paper MHC major histocompatibility complex - MLR mixed lymphocyte reactions - SSC saline sodium citrate - kbp kilobase pairs - SDS sodium dodecyl sulfate     

Restriction fragment length polymorphism of the major histocompatibility complex of the pig

Human HLA cDNA probes were used to analyze the restriction fragment length polymorphism (RFLP) of the SLA major histocompatibility complex in swine. Cellular genomic DNA from 19 SLA homozygous pigs representing 13 different haplotypes was digested with restriction endonucleases Eco RI, Hind III, or Bam H1, separated by electrophoresis, and transferred onto diazobenzyloxymethyl paper by the Southern blot technique. The blots were probed with ³²P-labeled class I or beta-DR class II cDNA. Depending on the haplotypes and the endonucleases used, seven to ten restriction fragments hybridized with the class I probe, and five to seven with the beta-DR probe. Their sizes ranged from 3.4 to 22 kilobase-pairs. Few bands were common to all 13 haplotypes. With all but one haplotype, identical autoradiogram patterns were obtained from unrelated, but phenotypically SLA-identical pigs, suggesting that most of the RFLP revealed were controlled by the SLA region. Further polymorphism was found in a group of seven unrelated pigs which typed serologically as SLA A15 CI B18 homozygotes but could be divided into two subgroups, with five animals in one subgroup and two in the other, when the genomic DNA was hybridized with the class I probe. When the class 11 beta-DR probe was tested on the same seven pigs, another subdivision was seen, and this correlated with MLR data. These results demonstrate that HLA class I and class II probes can be used to identify certain well-established SLA haplotypes and to identify subclasses within at least one SLA haplotype.Abbreviations MHC major histocompatibility complex - MLR mixed lymphocyte reaction - kbp kilobase pair(s) - RFLP restriction fragment length polymorphism     

The analyses of antigen and DNA polymorphism within the bovine major histocompatibility complex: 1. The class I antigens

Summary. Serology, isoelectric focusing (IEF) of expressed antigens, and restriction fragment length polymorphisms (RFLP) were compared for the identification of BoLA class I haplotypes. Expressed antigens identified as bands by IEF correlated well with serological definition confirming and extending our earlier findings (Joosten et al. 1988). Comparison of serology and isoelectric focusing bands with restriction fragments was more complicated; fragments were found which correlated both with broadly reacting and antigen specific sera. We also found correlation of fragments with two or more sera which showed no cross-reactivity. Fragments unique to particular haplotypes were also observed.
Serology remains the simplest method of typing BoLA class I antigens. Isoelectric focusing generally agrees with serological definition of antigens and detects antigens not yet defined by serology. It may also be useful in defining the products of other expressed BoLA class I loci. In order to identify RFLPs which could be used for typing, comparison with serology or IEF is essential. Haplotype specific RFLPs could be useful in identifying genes linked to the MHC.     

The analyses of antigen and DNA polymorphism within the bovine major histocompatibility complex: 1. The class I antigens

Serology, isoelectric focusing (IEF) of expressed antigens, and restriction fragment length polymorphisms (RFLP) were compared for the identification of BoLA class I haplotypes. Expressed antigens identified as bands by IEF correlated well with serological definition confirming and extending our earlier findings (Joosten et al. 1988). Comparison of serology and isoelectric focusing bands with restriction fragments was more complicated; fragments were found which correlated both with broadly reacting and antigen specific sera. We also found correlation of fragments with two or more sera which showed no cross-reactivity. Fragments unique to particular haplotypes were also observed. Serology remains the simplest method of typing BoLA class I antigens. Isoelectric focusing generally agrees with serological definition of antigens and detects antigens not yet defined by serology. It may also be useful in defining the products of other expressed BoLA class I loci. In order to identify RFLPs which could be used for typing, comparison with serology or IEF is essential. Haplotype specific RFLPs could be useful in identifying genes linked to the MHC.     

An association between a lymphocyte antigen in sheep and the response to vaccination against the parasite Trichostrongylus colubriformis

Lymphocyte antigens were tested in sheep which had been selected for responsiveness to vaccination against the intestinal nematode Trichostrongylus colubriformis. These sheep had been bred in an assortative mating programme which produced offspring designated as either “high responders” or “low responders”, with highly heritable resistance or susceptibility.Ovine lymphocyte antigen (OLA) typing antisera were obtained from parous ewes in the course of matings which produced the high and low responder flocks. A particular antigen (SY1) was found to be present in high frequency on the lymphocytes of high responder (72·2%) and in lower frequency (21·9%) on the lymphocytes of low responder rams. In ewes, the frequency for high responders was 65·7% and for low responders it was 33·5%. A similar association between the SY1 antigen and low faecal egg count was found in random-bred sheep which had been vaccinated with irradiated larvae and challenged with normal larvae. The conclusion was drawn that this lymphocyte antigen was likely to be part of the sheep major histocompatibility complex which influenced the immune response of sheep to vaccination against the parasite.     

Major histocompatibility complex (MHC) related cDNA probes associated with antibody response in meat-type chickens

The major histocompatibility complex (MHC) region was examined as a set of candidate genes for association between DNA markers and antibody response. Intercross F₂ families of chickens were generated from a cross between high (HC) and low (LC) Escherichia coli _i antibody lines. Restriction fragment length polymorphism (RFLP) analysis was conducted by using three MHC-related cDNA probes: chicken MHC class IV ( B-G ), chicken MHC class I ( B-F ), and human MHC-linked Tap2 . Association between RFLP bands and three antibody response traits ( E. coli , sheep red blood cells and Newcastle disease virus) were determined by two methods: by statistically analyzing each band separately and also by analyzing all bands obtained from the three probes by using multiple regression analysis to account for the multiple comparisons. The MHC class IV probe was the highest in polymorphisms but had the lowest number of bands associated with antibody response. The MHC class I probe yielded 15 polymorphic bands of which four exhibited association with antibody response traits. The Tap2 probe yielded 20 different RFLP bands of which five were associated with antibody production. Some Tap2 bands were associated with multiple antibody response traits. The multiband analysis of the three probes' bands revealed more significant effects than the analysis of each band separately. This study illustrates the efficacy of using multiple MHC region probes as candidate markers for quantitative trait loci (QTLs) controlling antibody response in chickens.     

Molecular genetic analysis of the major histocompatibility complex in an ELA typed horse family

Restriction fragment length polymorphism was studied in an ELA typed horse family which included a stallion, a mare with two full-sibs, another mare with three full-sibs and, in addition, three paternal half-sibs. DNA samples from all individuals were investigated by Southern blot analysis using three restriction enzymes (EcoRI, HindIII or TaqI) and human cDNA class I, class II (DR beta) and class III (C4) probes. In addition, a genomic class II DQ alpha probe was used. Fragments hybridized with the various probes revealed the existence of DNA sequences homologous to HLA class I, DR beta, DQ alpha and C4 genes in the horse. Polymorphic fragments were found when DNA was hybridized with class I and class II probes irrespective of the enzyme used; but hybridization with the C4 probe did not reveal variability. All polymorphic fragments segregated according to the ELA serological specificities, thus indicating a close linkage between the different revealed subregions. Banding patterns suggest that the horse possesses about 20-30 class I genes, probably more than one DR beta and DQ alpha genes and possibly only one C4 gene. The high degree of polymorphism observed suggests that molecular DNA typing may represent a potentially powerful aid to decision in parentage control determination.     

A biochemical characterization of feline MHC products: Unusually high expression of class 11 antigens on peripheral blood lymphocytes

The polymorphism of feline MHC antigens was examined using biochemical methods. The following observations were made: (1) feline class I and II antigens are polymorphic. Their biochemical features were established using rabbit and mouse reagents directed against human MHC products; they resemble those observed for other mammalian species; (2) the expression of class II antigens in unstimulated cat peripheral blood lymphocytes (PBLs) appears to be unusually high. Cat PBLs express far more class II than class I antigens, whereas in human Epstein-Barr virus-transformed lines, which are known to express relatively large amounts of class II antigens, the situation is reversed.Abbreviations used in this paper EBV Epstein-Barr virus - FLA feline lymphocyte antigen - MHC major histocompatibility complex - MLC mixed lymphocyte culture - MLR mixed lymphocyte reaction - PBL peripheral blood lymphocyte - RT room temperature - TX-114 Triton X-114 - 1-D IEF one-dimensional isoclectric focusing - 2-D SDS-PAGE twodimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis     

Restriction fragment length polymorphism of DQB and DRB class II genes of the ovine major histocompatibility complex

The ovine major histocompatibility complex (MhcOvar) class II region was investigated by Southern blot hybridizations using ovine probes specific for the second exons of Ovar-DRB and Ovar-DQB genes. Multiple bands were revealed when genomic DNA was digested with each of five restriction enzymes (Bam HI, Eco RI, Hin dIII, PvuII and TaqI), and successively hybridized with the two radiolabeled ovine probes. Restriction fragment length polymorphisms (RFLPs) were analysed in 89 sheep originating from six inbred families and the inheritance of the fragment patterns was determined. Forty-one fragments were recorded with the DQB probe; 32 were detected with the DRB probe. They constituted 9 DQB and 10 DRB allelic patterns. Twelve DQB-DRB haplotypes were resolved in this study.     

Analysis of the equine lymphocyte antigen system by Southern blot hybridization

Fourteen Standardbred horses homozygous for one of six equine lymphocyte antigen (ELA) specificities (A1, A3, A4, A5, A6, or A10) were analyzed by Southern blot hybridization using DNA probes derived from the mouse major histocompatibility complex (MHC). Total genomic DNA from peripheral lymphocytes was digested with the restriction enzymes Hind III, Pvu II, or Eco RI. Twenty-three to thirty-three bands were generated for individual horses with the class I cDNA probe. The resulting band patterns revealed 12-14 nonpolymorphic fragments, which is consistent with the highly conservedQa/Tla genes seen in other species. The remaining 10–19 bands displayed significant polymorphism; no two animals had identical band patterns when studied with all three enzymes. The polymorpism was markedly decreased between animals of the same ELA serotypes. Unique bands were identified in both Al horses and all four A6 animals. Pvu II digestions of lymphocyte DNA were hybridized with mouse MHC class II probes. A cDNA probe for theE gene revealed only a single nonpolymorphic band. In contrast, a cDNA probe for theH-2 A locus displayed three to five strong bands in each animal with polymorphism that was most pronounced between horses of different ELA serotypes. Genomic DNA probes forAandE genes both revealed multiple polymorphic bands. However, cross-hybridization between these two probes prevented distinction betweenA andE equivalent loci. The reduced polymorphism evident within ELA specificities is consistent with the concept that the equine lymphocyte antigen system includes two families of closely linked MHC genes.     

Identification of equine major histocompatibility complex haplotypes using polymorphic microsatellites

A system for identifying equine major histocompatibility complex (MHC) haplotypes was developed based on five polymorphic microsatellites located within the MHC region on ECA 20. Molecular signatures for 50 microsatellite haplotypes were recognized from typing 353 horses. Of these, 23 microsatellite haplotypes were associated with 12 established equine leucocyte antigen (ELA) haplotypes in Thoroughbreds and Standardbreds. Five ELA serotypes were associated with multiple microsatellite subhaplotypes, expanding the estimates of diversity in the equine MHC. The strong correlations between serological and microsatellite typing demonstrated a linkage to known MHC class I protein polymorphisms and validated this assay as a useful supplement to ELA serotyping, and in some applications, a feasible alternative method for MHC genotyping in horse families and in population studies.